The present invention relates to proteolytic enzymes obtainable from hyperthermophilic bacteria and processes for producing them.
Hyperthermophilic bacteria, i.e. bacteria which thrive on temperatures around the boiling point of water, are found in the ocean close to geothermal springs. Since these bacteria live in high temperature environments, their enzymes which are essential to sustaining life, such as digestion and respiration, must be able to function at such extreme temperature conditions. Enzymes in common mesophilic bacteria (i.e., an organism that can grow at intermediate temperatures compared to the upper and lower extremes for all organisms) degenerate rapidly at such high temperatures.
Proteins which can function at high temperatures can be extremely advantageous for use in a number of industries. For example, soda syrup, laundry detergent, and many pharmaceuticals contain, or are manufactured by, enzymes extracted from bacteria. If enzymes from hyperthermophilic bacteria were used in place of the enzymes commonly used today, the processes to make these products could be performed at higher temperatures. Higher temperatures speed up reactions and prevent contamination by fungi and common bacteria. Alternatively, lesser amounts of the enzymes from hyperthermophilic bacteria might be required to sustain enzymatic processes under current temperature conditions, where the thermostability of such enzymes correlates with a longer useful life under those conditions.
A number of microorganisms capable of growth at or above 100.degree. C. (i.e., hyperthermophiles) have been isolated from several terrestrial and marine environments and are of considerable scientific interest. (See Kelly, R. M., and J. W. Deming, 1988, "Extremely Thermophilic Archaebacteria: Biological and Engineering Considerations", Chem. Engr. Prog. 4:47-62; and Wiegel J., and L. G. Ljungdahl, 1986, "The Importance of Thermophilic Bacteria In Biotechnology," CRC Crit. Rev. Microbiol. 3:39-107.) However, it has not previously been possible to take full advantage of the utility potential of these organisms, in large part, because of a lack of understanding of their growth and metabolic characteristics.
Detailed study of specific enzymes from hyperthermophiles is just beginning, and the few known reports on such enzymes that have been published so far all appeared in 1989. For example, it has been shown (Pihl, T. D. et al., 1989, Proc. Natl. Acad. Sci. 86:138-141) that an extremely thermostable hydrogenase isolated from Pyrodictium brockii is immunologically related to the comparable enzyme in the Bradyrhizobium japonicum, a mesophile. Adams and coworkers have described several distinctive characteristics of a hydrogenase (Bryant, F. O. & Adams, M. W. W., 1989, J. Biol. Chem. 264:5070-5079) and a ferredoxin (Aono, S., et al., 1989, J. Bacteriol. 171:3433-3439) from the hyperthermophilic bacterium, Pyrococcus furiosus.
Proteases are important physiologically in protein digestion and turnover within a cell and industrially for degrading various proteinaceous materials [Kalisz, H. M., 1988, In Advances in Biochemical Engineering/Biotechnology Vol. 36 (Ed. Fiechter, A.), 1-65, Springer-Verlag, Berlin and Heidelberg]. Serine proteases, which includes subtilisin-like proteases and proteinase K, show unusual resistance to denaturation by urea, guanidinium chloride, sodium dodecyl sulfate (SDS), and other detergents [Weber, K., Pringle, J. R. & Osborn, M., 1972, In Methods in Enzymology Vol. 26, Part C (Ed. C. H. W. Hirs & S. N. Timasheff), 3-27 (Academic Press, New York and London]. For example, Deane et al.(1987, J. Gen. Microbiol. 133:2295-2301) reported an SDS-resistant protease from Vibrio alginolyticus which is active at 37.degree. C. and alkaline pH, and through inhibitor studies, categorized it as a serine protease (Deane, S. M., et at., 1987, J. Gen. Microbiol. 133:391-398). The approximate molecular weight of this protease was 54,000 daltons based on electrophoresis in SDS, and it was active in the presence of SDS at 37.degree. C. Active fragments (with MW's of 41,000 and 37,000 daltons) of the protein formed when it was dialyzed against distilled water.
Some proteases have been reported that show unusual thermostability. For example, Cowan et al. (1987, Biochem, J. 247:121-133) recently described the discovery of an extracellular protease from a Desulfurococcus species which has a half-life of seventy to ninety minutes at 95.degree. C, but denatures rapidly above 100.degree. C. It is believed that this represents the most extreme thermostability yet reported for a proteolytic activity. Other similarly thermostable proteases, primarily from Thermus species, have also been described (Cowan, D. A. & Daniel, R. M., 1982, Biochim. Biophys. Acta 705:293-305; Khoo, T. C., et al., 1984, Biochem. J. 221:407-413; Taguchi, H., et al., 1983, J. Biochem. 93:7-13; Matsuzawa, H., et al., 1983, O. Agric. Biol. Chem. 47:25-28). Proteases from hyperthermophilic bacteria, however, do not appear to be known in the art.
Accordingly, a major object of the present invention is to provide methods for producing proteolytic enzyme preparations from hyperthermophilic bacteria such as P. furiosus.
It is also an object of the present invention to provide purified proteolytic enzyme preparations exhibiting thermostable activities that are resistant to detergents and thus are useful for various industrial applications.